Unrestrained Guinea Pigs Research Articles

An inexpensive system for evaluating the tussive and anti-tussive properties of chemicals in conscious, unrestrained guinea pigs

Introduction: Commercial whole-body plethysmography systems used to evaluate the anti-tussive potential of drugs employ sophisticated technology, but these systems may be cost prohibitive for some laboratories. The present study describes an alternative, inexpensive system for evaluating the tussive and anti-tussive potential of drugs in conscious, unrestrained guinea pigs. Methods: The system is composed of a transparent small animal anesthesia induction box fitted with a microphone, a camera and a pneumotachometer to simultaneously capture audio, video, air flow and air pressure in real time. Data acquisition and analysis was performed using free software for audio and video, and a research pneumotach system for flow and pressure. System suitability testing was performed by exposing conscious, unrestrained guinea pigs to nebulized aqueous solutions of a selective agonist for TRPV1 (citric acid) or a selective agonist for TRPA1 (AITC), with or without pre-treatment with a selective antagonist for TRPV1 (BCTC) or a selective antagonist for TRPA1 (HC-030031). Results: The system easily discerned coughs from other respiratory events like sneezes. System suitability test results are as follows: AITC caused 10.7 (SEM=1.4592) coughs vs. 5.8 (SEM=1.6553) when pre-treated with HC-030031 (P<0.05). Citric acid caused 12.4 (SEM=1.4697) coughs vs. 3.2 (SEM=1.3928) when pre-treated with BCTC (P<0.002). Discussion: We have described in detail an inexpensive system for evaluating the tussive and anti-tussive potential of chemicals in conscious, unrestrained guinea pigs. Suitability testing indicates that the system is comparable to a commercial whole-body plethysmography system for detecting and differentiating between coughs and sneezes. This system may provide some investigators a cost-conscious alternative to more expensive commercial whole-body plethysmography systems.

Rosuvastatin Blocks hERG Current and Prolongs Cardiac Repolarization

Blocking of the potassium current I(Kr) [human ether-a-go-go related gene (hERG)] is generally associated with an increased risk of long QT syndrome (LQTS). The 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitor, rosuvastatin, is a methanesulfonamide derivative, which shows structural similarities with several I(Kr) blockers. Hence, we assessed the effects of rosuvastatin on cardiac repolarization by using in vitro, ex vivo, and in vivo models. Patch clamp experiments on hERG-transfected human embryonic kidney (HEK) 293 cells established the potency of rosuvastatin to block hERG [half maximal inhibitory concentration (IC(50) ) = 195 nM]. We showed in isolated guinea pig hearts that 195 nM rosuvastatin prolonged (basic cycle length of 250 ms; p < 0.05) the monophasic action potential duration at 90% repolarization (MAPD(90) ) by 11 ± 1 ms. Finally, rosuvastatin (10 mg/kg, intraperitoneal) prolonged corrected QT interval (QTc) in conscious and unrestrained guinea pigs from 201 ± 1 to 210 ± 2 ms (p < 0.05). Thus, rosuvastatin blocks I(Kr) and prolongs cardiac repolarization. In additional experiments, we also show that hERG blockade in HEK 293 cells was modulated by coexpression of efflux [breast cancer resistance protein (BCRP), multidrug resistance gene (MDR1)] and influx [organic anion transporting polypeptide (OATP) 2B1] transporters involved in the disposition and cardiac distribution of the drug. Genetic polymorphisms observed for BCRP, MDR1, and OATP2B1, and IC(50) determined for hERG blocking lead us to propose that some patients may be at risk of rosuvastatin-induced LQTS.

Does the sedative agent, JM-1232(−) cause QT prolongation with subsequent torsades de pointes?

Aims JM-1232(−) ((−)-3-[2-(4-methyl-1-piperazinyl)-2-oxoethyl]-2-phenyl-3,5,6,7-tetrahydrocyclopenta[f] isoindole-1(2H)-one) is a novel isoindoline compound that acts on benzodiazepine receptors. Clinical trials are being conducted to assess its potential clinical use as an intravenous anesthetic or sedative agent. In the present study, we investigated the possibility of QT prolongation caused by JM-1232(−). Main methods We examined telemetry electrocardiogram (ECG) recordings from unrestrained guinea pigs after intravenous administration of 1, 10, 25 or 50 mg/kg of JM-1232(−). We also examined in situ measurements of monophasic action potential duration at 30 and 90% of repolarization (MAP 30 and MAP 90, respectively) and MAP triangulation (MAP 90–30) from guinea pig hearts that were perfused on a Langendorff apparatus with 0, 0.1, 1, 5, 10 and 20 μg/ml of JM-1232(−). The telemetry ECG recordings showed QTc prolongation 20 min after administration of JM-1232(−) at 50 mg/kg. Key findings In Langendorff-perfused hearts, MAP 30, which indicates Ca 2+ current, was significantly shortened after perfusion with JM-1232(−) at 10 and 20 μg/ml. MAP 90, which indicates K + current, was significantly prolonged after perfusion with JM-1232(−) at 20 μg/ml; therefore, this prolongation was attributed to a decreased heart rate. MAP triangulation (MAP 90–30) was significantly prolonged after perfusion with JM-1232(−) at 5, 10 and 20 μg/ml; however, this prolongation was attributed to the shortened MAP 30, suggesting a possible inhibition of Ca 2+ influx. Significance High-dose (more than 50 times the hypnotic dose) JM-1232(−) treatment associated with a high plasma concentration of the drug is unlikely to cause QT prolongation with subsequent torsades de pointes.

Effects of repeated injections of fibroblast‐stimulating lipopeptide‐1 on fever, formation of cytokines, and on the responsiveness to endotoxin in guinea‐pigs

We investigated, whether the Toll-like receptors (TLRs)-2/6-agonist fibroblast-stimulating lipopeptide-1 (FSL-1), like the TLR-4 agonist lipopolysaccharide (LPS), induces a state of tolerance. We further tested the influence of repeated pre-treatment with FSL-1 on the animals' responsiveness to LPS. Abdominal temperature was recorded in unrestrained guinea-pigs with intra-abdominally implanted radiotransmitters. Circulating concentrations of tumour necrosis factor (TNF) and interleukin-6 (IL-6) were measured with specific bioassays. We tested the effects of intra-arterial (i.a.) or intraperitoneal (i.p.) injections of 100 microg kg(-1) FSL-1, repeated five times at intervals of 3 days. The animals' responses to i.a. or i.p. injections of 10 microg kg(-1) LPS were determined another 3 days later and compared to those of naïve guinea-pigs. The FSL-1-induced TNF peak was significantly attenuated starting with the third i.a. administration, while fever was unimpaired and the IL-6-peak just tended to decrease. Fever and IL-6 in response to i.a. injections of LPS were identical in both groups, while circulating TNF was higher in naïve compared to FSL-1 pre-treated animals. The effects of repeated i.p. injections of FSL-1 were more pronounced resulting in attenuation of fever as well as circulating TNF and IL-6, the strongest reduction observed after the third stimulation with FSL-1. Repeated i.p. pre-treatment with FSL-1 induced hyporesponsiveness to i.p. administration of LPS compared to naïve animals with regard to fever and especially with regard to LPS-induced formation of cytokines. There is a development of tolerance to FSL-1 and cross-tolerance between FSL-1 and LPS depending on the route of administration of the respective TLR-2/6 and TLR-4 agonists.

CONTINUOUS INFUSION OF SIVELESTAT SODIUM HYDRATE PREVENTS LIPOPOLYSACCHARIDE‐INDUCED INTESTINAL PARALYSIS AND HYPOTENSION IN CONSCIOUS GUINEA‐PIGS

1. Sivelestat sodium hydrate (sivelestat), a neutrophil elastase inhibitor, is used to treat acute lung injury associated with systemic inflammatory response syndrome, but its effects have not been described for endotoxaemia. In the present study, we examined the effects of a continuous infusion of sivelestat on intestinal mechanical activity and blood pressure using an endotoxaemic model in conscious, unrestrained guinea-pigs. 2. Guinea-pigs underwent laparotomy while anaesthetized and were implanted with a force transducer sutured onto the taenia caecum. With this transducer, changes in tension in the intestinal longitudinal muscle were measured continuously via telemetry. Catheters were inserted into the carotid artery and jugular vein, were tunnelled subcutaneously and were accessed from the back of the neck. These catheters were connected to a cannula swivel and were used to monitor arterial pressure as well as to administer drugs i.v. in conscious, unrestrained guinea-pigs. Twenty hours after surgery, guinea-pigs received a single dose of lipopolysaccharide (LPS; 0.3 mg/kg, i.p.) 10 min after the start of a continuous 2 h i.v. infusion of sivelestat (30 mg/kg per h) or vehicle (saline). Elastase activity before and after sivelestat or vehicle administration was measured spectrometrically using a specific synthetic substrate. 3. We confirmed that intestinal longitudinal muscle tension decreased 2-3 h after LPS administration in the control group, with a concurrent decline in blood pressure. In guinea-pigs treated with sivelestat, the LPS-induced decreases in muscle tension and blood pressure were significantly reduced. In LPS-treated control guinea-pigs, serum elastase activity was elevated and this increase was significantly attenuated by administration of sivelestat. 4. The findings from the present study suggest that sivelstat can effectively reduce intestinal dysfunction and attenuate LPS-induced decreases in blood pressure in endotoxaemia.

Inhibition of histamine-induced bronchospasm in guinea pigs treated with Cecropia glaziovi Sneth and correlation with the in vitro activity in tracheal muscles

A standardized aqueous extract (AE) and a purified fraction (BuF) of Cecropia glaziovi Sneth leaves were tested in unrestrained guinea pigs challenged with histamine. Changes of the respiratory pressure and rate were recorded in a whole body plethysmograph before and after treatment. The concentration of histamine necessary to produce bronchospasm was increased by five-fold following administration of AE (1.0 g/kg p.o.), and by two-fold after treatment with the semi-purified procyanidin/flavonoids enriched BuF (0.1 g/kg p.o.). Both effects were blocked by previous treatment with propranolol (10.0 mg/kg i.p.). In vitro incubation of BuF (0.1-1.0 mg/ml) decreased by 13-55% the maximal response of guinea pig tracheal muscle to histamine, without significant change of EC50. The results confirmed old reports on the useful pulmonary effects of Cecropia extracts. The bronchodilation observed in vivo seems to be related to beta-adrenergic activity observed in vitro only with high concentrations of the purified extract.

Mechanisms of Capsaicin- and Citric-Acid-Induced Cough Reflexes in Guinea Pigs

Previously, we developed a novel coughing model that evoked coughs via citric acid microinjection into the larynx of unanesthetized, unrestrained guinea pigs. Here, we compared the effects of capsaicin and citric acid administration into the larynx using this model. Inhalation of capsaicin (30 µM) or citric acid (0.4 M) for 5 min induced cough mimetic responses over a 10-min observation period (mean ± S.E.M.: 8.85 ± 1.60 and 10.40 ± 1.17 coughs, respectively, n = 18). Microinjection of 0.4 M citric acid into the larynx (20 µl in total in 10 aliquots of 2 µl at 30-s intervals) induced 27.29 ± 2.69 coughs over a 10-min observation period, whereas microinjection of 3 mM capsaicin evoked a maximum of 8.32 ± 1.82 coughs (n = 18). Desensitization of C-fibers by capsaicin (100 mg/kg, s.c.) abolished the coughs evoked upon inhalation or microinjection of capsaicin and inhalation of citric acid, but had no significant effect on those induced by microinjection of citric acid. These results indicate that there were fewer nonmyelinated C-fiber afferents in the larynx area than in other regions (bronchi or alveoli). Consequently, citric acid microinjected into the larynx may induce the cough reflex by stimulating other fibers (for example, Aδ-fibers), rather than C-fibers. In contrast, inhaled citric acid predominantly stimulated C-fibers, and hardly affected Aδ-fibers.

Magnetocardiography for pharmacology safety studies requiring high patient throughput and reliability

Recent guideline drafts of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) underline the necessity to test nonantiarrhythmic drugs for their potential to prolong the QT or the corrected QT (QTc) interval. The implementation of these guidelines requires a large amount of ECG measurements on animals and humans in preclinical and clinical phases of the drug development process. We propose the use of magnetocardiography (MCG) as a complementary method with particular advantages in high-throughput studies, where signal quality and reliability are key factors. Our proposal is based on a review of recent MCG studies investigating the repolarization phase and results of methodological work assessing QT interval parameters from the MCG. The applicability of MCG for pre-clinical in-vivo studies is demonstrated by the ease of measurement in unrestrained non-anesthetized rabbits, guinea pigs, and hamsters..

Cough reflex induced by microinjection of citric acid into the larynx of guinea pigs: new coughing model.

We developed a new coughing model that evoked coughs by microinjection of citric acid into the larynx in unanesthetized unrestrained guinea pigs; additionally, we recorded synchronous sounds and waveforms of coughing utilizing built-in microphones and a whole body plethysmograph. The coughing model was able to distinguish a coughing response from other expiratory responses, such as an expiratory reflex or a sigh, by examining the waveform of the expiratory response and the existence of sound. It was not necessary to distinguish a cough from a sneeze, since the administration site was restricted to the larynx. Microinjection of 0.4 M citric acid, total of 20 microl (10 times, 2 microl at 30-s intervals), induced coughs (27.03 +/- 4.03 coughs in 10-min observation) that were stable and independent of the inhalation volume. In the inhalation studies, animals were exposed to citric acid only once, because the number of coughs remarkably decreased with repeated administration at intervals of 24 h (tachyphylaxis). However our coughing model was able to repeatedly challenge the microinjection of citric acid at an interval of 24 h. These results indicated that this coughing model was highly sensitive and correctly assessed the cough response.

Power spectral analysis of heart rate variability for assessment of diurnal variation of autonomic nervous activity in guinea pigs.

We established characteristics of power spectral analysis of heart rate variability, and assessed the diurnal variations of autonomic nervous function in guinea pigs. For this purpose, an electrocardiogram (ECG) was recorded for 24 hr from conscious and unrestrained guinea pigs using a telemetry system. There were two major spectral components, at low frequency (LF) and high frequency (HF) bands, in the power spectrum of HR variability. On the basis of these data, we defined two frequency bands of interest: LF (0.07-0.7 Hz) and HF (0.7-3.0 Hz). The power of LF was higher than that of HF in the normal guinea pigs. Atropine significantly reduced power at HF. Propranolol also significantly reduced power at LF. Furthermore, the decrease in the parasympathetic mechanism produced by atropine was reflected in a slight increase in the LF/HF ratio. The LF/HF ratio appeared to follow the reductions of sympathetic activity produced by propranolol. Autonomic blockade studies indicated that the HF component reflected parasympathetic activity and the LF/HF ratio seemed to be a convenient index of autonomic balance. Nocturnal patterns, in which the values of heart rate in the dark phase (20:00-06:00) were higher than those in the light phase (06:00-20:00), were observed. However, the HF, LF and the LF/HF ratio showed no daily pattern. These results suggest that the autonomic nervous function in guinea pigs has no clear circadian rhythmicity. Therefore, this information may be useful for future studies concerning the autonomic nervous function in this species.

Coughing model by microinjection of citric acid into the larynx in guinea pig

Many studies of cough were performed under the restrained or anesthetized condition, and coughs were evoked by inhalation of capsaicin or citric acid. Inhalation of irritants induced by "diving response" with apnea and coughs, and these responses induced a change of tidal volume. As a result, respiratory responses are dependent on the inhalation volume. Therefore we developed a new coughing model, and coughs were evoked by microinjection of citric acid into the larynx in the unanesthetized unrestrained guinea pig. Microinjection of 7.5% citric acid (2 microliters/30 s, 5 min) induced coughs (27.03 +/- 4.03 coughs/10 min), and citric acid-induced responses were stable independent of the inhalation volume. In the inhalation studies, animals were exposed to citric acid only once because induced-responses were remarkably decreased by repeated administration at an interval of 24 h. However in our coughing model it was possible to repeatedly challenge the animals by microinjection of citric acid at intervals of 24 h. Microinjection of citric acid into the larynx induced coughs in Sprague-Dawley rats, but inhalation of citric acid did not induce cough. These results indicate that this coughing model is highly sensitive and correctly assessed cough responses.

The daily pattern of heart rate, body temperature, and locomotor activity in guinea pigs.

We studied the characteristics of the rhythmicity of heart rate (HR), body temperature (BT), and locomotor activity (LA) in conscious and unrestrained guinea pigs using a telemetry system. HR and/or LA in some guinea pigs clearly showed circadian rhythms, but in others there were no significant daily patterns; BT did not show significant daily rhythms. These results suggest that guinea pigs might have different individual characteristics of rhythmicity, and we should, therefore, be careful when using guinea pigs in chrono-biomedical research. We believe that the results of this study may be useful for future biomedical studies using guinea pigs.

Identification of mechanisms involved in the acute airway toxicity induced by parathion

Organophosphates are still widely used worldwide and cause thousands of intoxications every year. In this work we investigated the mechanisms of parathion (Pth) airway toxicity, using biochemical and functional approaches. A plethysmographic technique for unrestrained guinea pigs was used to analyze Pth-induced modifications of airway mechanics and responsiveness to acetylcholine (ACh: 0.1-3.2 mg/ml, 2-min inhalation each dose). The isolated perfused rabbit lung preparation was used to study the acute effects of Pth on airway responsiveness to ACh (10(-8)-10(-3) M), histamine (10(-8)-10(-3) M) and substance P (10(-10)-10(-6) M), pulmonary acetylcholinesterase inhibition and cytochrome P450 (P450) activity, and their modifications with previous administration of Pth (1 mg/kg s.c. daily, 7 days). We found that: (1) In guinea pigs Pth (3.2-17 mg/kg i.p.) produced a dose-dependent increase in a lung resistance index (iRL), which was greatly reverted (approximately 50%) by salbutamol (2 mg/ml, 2-min inhalation, or 10 microg/kg i.p.). This salbutamol effect was transient (5-10 min), suggesting that this bronchodilator triggered additional obstructive mechanisms. (2) Pth increased the water content in lung parenchyma samples, but not in trachea or bronchi, and augmented the respiratory secretions measured through monosaccharide content in bronchoalveolar lavage. (3) The increase in iRL was greater in female animals, probably due to a higher P450 basal activity, and completely blocked by pharmacological inhibition of P450 with piperonyl butoxide (500 mg/kg i.p.). (4) In male guinea pigs a subclinical dose of Pth (10 mg/kg i.p.) induced airway hyperresponsiveness to ACh. In isolated perfused rabbit lung Pth (10(-6) M) produced airway hyperresponsiveness to ACh and histamine, the latter prevented by atropine (10(-5) M). (5) Repetitive exposure to subclinical doses (1 mg/kg s.c.) of Pth during 1 week caused approximately 80% inhibition of P450 activity in rabbits, which was not enough, however, to prevent the functional manifestation of Pth toxicity in the airways.

Role of tachykinin NK1 and NK2 receptors in allergen-induced early and late asthmatic reactions, airway hyperresponsiveness, and airway inflammation in conscious, unrestrained guinea pigs.

Using a guinea pig model of allergic asthma, we investigated the effects of the inhaled, highly selective nonpeptide tachykinin NK1 and NK2 receptor antagonists SR 140333 and SR 48968, respectively, on allergen-induced early (EAR) and late (LAR) asthmatic reactions, airway hyperreactivity (AHR) after these reactions, and infiltration of inflammatory cells in the airways. Both SR 140333 (100 nM, 3 min) and SR 48968 (100 nM, 3 min) had no effect on the severity of the EAR, while the NK2 receptor antagonist SR 48968, but not the NK1 receptor antagonist SR 140333, caused significant inhibition of the LAR. SR 140333 significantly reduced the allergen-induced AHR to histamine, both after the EAR and the LAR. By contrast, SR 48968 did not affect the AHR after the EAR, but significantly attenuated the AHR after the LAR. Bronchoalveolar lavage studies performed after the LAR indicated that SR 140333 caused significant inhibition of allergen-induced infiltration of eosinophils, neutrophils and lymphocytes, while SR 48968 attenuated the infiltration of neutrophils and lymphocytes, but not of eosinophils. Both NK receptor antagonists tended to reduce the accumulation of ciliated epithelial cells in the airways. These results indicate that NK1 and NK2 receptors are importantly, but differentially, involved in the development of allergen-induced airways obstruction, AHR and infiltration of inflammatory cells in the airways. Therefore, both NK1 and NK2 receptor antagonists, or dual NK1 and NK2 antagonists, could be useful in the treatment of allergic asthma.

Role of tachykinin NK2-receptor activation in the allergen-induced late asthmatic reaction, airway hyperreactivity and airway inflammatory cell influx in conscious, unrestrained guinea-pigs.

1. In a guinea-pig model of allergic asthma, we investigated the involvement of the tachykinin NK2 receptors in allergen-induced early (EAR) and late (LAR) asthmatic reactions, airway hyperreactivity (AHR) after these reactions and inflammatory cell influx in the airways, using the selective non-peptide NK2 receptor antagonist SR48968. 2. On two different occasions, separated by a 1 week interval, ovalbumin (OA)-sensitized guinea-pigs inhaled either vehicle (3 min) or SR48968 (100 nM, 3 min) at 30 min before as well as at 5.5 h after OA provocation (between the EAR and LAR) in a random crossover design. 3. SR48968 had no significant effect on the EAR, but significantly attenuated the LAR by 44.2+/-16.4% (P<0.05) compared to saline control. 4. The NK2 receptor antagonist did not affect the OA-induced AHR to histamine after the EAR at 5 h after OA challenge (3.59+/-0.59 fold increase in histamine reactivity vs 3.79+/-0.61 fold increase in the controls, NS), but significantly reduced the AHR after the LAR at 23 h after OA challenge (1.59+/-0.24 fold increase vs 1.93+/-0.15 fold increase, respectively, P<0.05). 5. Bronchoalveolar lavage studies performed at 25 h after the second OA provocation showed that SR48968 significantly inhibited the allergen-induced infiltration of neutrophils (P<0.05) and lymphocytes (P<0.01) in the airways. 6. These results indicate that NK2 receptor activation is importantly involved in the development of the allergen-induced late (but not early) asthmatic reaction and late (but not early) AHR to histamine, and that NK2 receptor-mediated infiltration of neutrophils and lymphocytes in the airways may contribute to these effects.

Dual action of iNOS-derived nitric oxide in allergen-induced airway hyperreactivity in conscious, unrestrained guinea pigs.

Using a guinea pig model of acute allergic asthma, we recently established that a deficiency of nitric oxide (NO) contributes to airway hyperreactivity (AHR) after the early asthmatic reaction (EAR) and that restoration of NO activity may contribute to the (partial) reversal of AHR after the late asthmatic reaction (LAR). In the present study, we investigated the role of iNOS-derived NO in the regulation of AHR to histamine after the LAR. Inhalation of a selective dose of the specific iNOS inhibitor aminoguanidine (0.1 mM, 3 min) had no effect on basal airway reactivity to histamine in unchallenged, ovalbumin-sensitized animals and did not affect the allergen-induced AHR after the EAR. By contrast, this dose of aminoguanidine significantly potentiated the partially reduced AHR after the LAR to the level of AHR observed after the EAR, indicating that induction of iNOS during the LAR contributes to the reversal of AHR. Inhalation of a higher aminoguanidine concentration (2.5 mM) shortly before the onset of the LAR diminished the AHR after the LAR and reduced the number of neutrophils, lymphocytes, and ciliated epithelial cells in the bronchoalveolar lavage at this time point. The results indicate that iNOS-derived NO may have both beneficial and detrimental effects on allergen-induced AHR to histamine after the LAR by functional antagonism of histamine-induced bronchoconstriction, and by promoting airway inflammation and epithelial damage on the other hand, respectively.

Guinea pig lung resistance shows circadian rhythmicity not influenced by ozone

Increased circadian variability of airway caliber is a key feature of asthmatic patients, but it has not been addressed in animal models of asthma. Furthermore, animal studies on circadian rhythmicity of airway resistance are very scanty. We used a plethysmographic method for unrestrained guinea pigs to monitor a lung resistance index (iRL) during 24 h. We found circadian variability of iRL values, which were fitted by a sinusoidal curve. Acrophase and bathyphase, characterizing the timing of narrowest and widest airway caliber, respectively, were found at 02:03, and 15:34 h. iRL values at these time-points were statistically different ( P<10 −5). Moreover, average resistance during the dark period was significantly higher ( P<0.0001) than during the light period. Immediately after an acute ozone exposure (3 ppm for 1 h) an increase in iRL was demonstrated ( P<0.01), which lasted for 2 h, and tended to remain high for the next hour. After guinea pigs recovered from this obstruction, the circadian rhythm and variability of airway caliber were unaffected. Our results show that a circadian rhythm of iRL takes place in guinea pigs, greatly resembling what occurs in humans, and that ozone exposure causes a transient airway obstruction, but fails to reproduce the increased variability of airway caliber observed in asthmatic patients.

Role of nitric oxide in the development and partial reversal of allergen-induced airway hyperreactivity in conscious, unrestrained guinea-pigs.

1. Using a conscious, unrestrained guinea-pig model of allergic asthma, we investigated the role of endogenous nitric oxide (NO) in the regulation of airway (hyper)reactivity to histamine before and after the allergen-induced early and late asthmatic reactions, by examining the effect of inhalation of the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME, 12 mM, 15 min) on the histamine-induced airway obstruction of ovalbumin-sensitized guinea-pigs before, and at 5.5 h and 23.5 h after allergen challenge. 2. Before allergen challenge, inhaled L-NAME caused a significant 2.02+/-0.25 fold increase (P<0.01) in airway reactivity to histamine; this effect was reversed within 2.5 to 6 h after administration. 3. After the allergen-induced early asthmatic reaction at 5 h after ovalbumin provocation, a significant 3.73+/-0.67 fold increase (P<0.01) of the airway reactivity to histamine was observed; subsequent inhalation of L-NAME at 5.5 h had no effect on the airway hyperreactivity, reassessed at 6 h. 4. After the late asthmatic reaction, at 23 h after ovalbumin provocation, a reduced, but still significant airway hyperreactivity to histamine (2.18+/-0.40 fold; P<0.05) was observed. Subsequent inhalation of L-NAME now significantly potentiated the partially reduced airway hyperreactivity 1.57+/-0.19 fold (P<0.05) to the level observed after the early asthmatic reaction. 5. When administered 30 min before allergen exposure, L-NAME significantly enhanced the allergen-induced early asthmatic reaction. However, when administered at 5.5 h after allergen provocation, L-NAME did not affect the subsequent late asthmatic reaction. 6. These results indicate that endogenous NO is involved the regulation of histamine- and allergen-induced bronchoconstriction and that a deficiency of cNOS-derived NO contributes to the allergen-induced airway hyperreactivity to histamine after the early asthmatic reaction, while a recovery of NO deficiency may account for the partial reversal of the allergen-induced airway hyperreactivity after the late asthmatic reaction.

Early Bronchoconstriction After Allergen Challenge of Nonanesthetized Guinea Pigs

Background: Bronchial asthma is a complex disease, which is characterized by spontaneous exacerbations of airway obstruction and persistent bronchial hyperresponsiveness. Animal models have fallen short of reproducing the human disease, particularly in mimicking the spontaneous and persistent airflow obstruction that characterized in asthma. In animals, airflow obstruction is usually assessed by measuring airflow resistance during tidal breathing under such invasive technique as tracheostomy and anesthesia. A noninvasive technique for measuring pulmonary function in small animals is needed to evaluate long-term changes in lung function during the course of experimentally produced disease without sacrificing the animal. Purpose: The purpose of this study was to evaluate early bronchoconstrcition after allergen challenge and airway responsiveness (AR) to inhaled methacholine in nonanethetized, unrestrained guinea pigs. Method: Guinea pig model of asthma was sensitized by subcutaneous injection with ovalbumin and challenged by inhalation of aerosolized ovalbumin(1% wt/vol ovlabumin). Airflow obstruction of conscious guinea pig was measured as specific airway resistance (airway resistance thoracic gas volume). Airway resistance and thoracic gas volume of conscious guinea pig were assessed by body plethysmography before challenge and at regular intervals for as long as 30 minutes after challenge. AR to aerosolized methacholine of asthma group was compared with that of control group in body plethysmography. Result: Asthma model developed in 13 (65%) among 20 guinea pigs, in which early responses occurred in the airways after the exposure to inhalation with ovalbumin. Airway challenge with ovalbumin caused increase in specific airway resistance, which peaked at 6 minutes and amounted to a increase from baseline. AR to aerosolized methacholine of asthma model increased significantly compared with control group. Conclusion: These results have showed a useful animal model to evaluate early bronchoconstrcition after allergen challenge and airway responsiveness in nonanethetized, unrestrained guinea pigs.

Determination of cortisol in guinea pig urine by high performance thin-layer chromatography, high performance liquid chromatography, and thin-layer chromatography/radioimmunoassay

In order to collect urinary samples from unrestrained guinea pigs, animals were kept in their familiar home cages with wood shavings for bedding. Cortisol was removed from shavings by a simple washing step, and an attempt was made to measure its concentrations by high performance thin-layer chromatography (HPTLC), high performance liquid chromatography (HPLC), or thin layer chromatography/radioimmunoassay (TLC-RIA). After intramuscular administration of 25 mg cortisol, cortisol excretion increased from about 20–30 μg/day to 400–500 μg/day (HPTLC: 531 μg/day, HPLC: 493 μg/day; TLC-RIA: 394 μg/day). Similarly, the treatment of the animals with 20 IU ACTH resulted in an augmented cortisol excretion, with mean values of 294 μg/day (HPTLC), 256 μg/day (HPLC) and 143 μg/day (TLC-RIA), respectively. The present study shows, for the first time, that cortisol excretion in unrestrained laboratory animals can be determined. Whilst the cortisol values measured by HPTLC and HPLC agree, the amounts measured by TLC-RIA were significantly lower. These differences are probably due to the presence of substances in urine or shavings which interfere with the radioimmunological determination. Hence, cortisol should be determined either by HPTLC or HPLC. Beside having a desirable specificity, these methods are more suited than TLC/RIA for steroid analysis since they confer the possibility of measuring additional steroids (e.g. precursors and/or metabolites of cortisol) in a single urine extract. This is especially the case for the HPTLC method since substances can be transformed into fluorescent derivatives.